The present invention relates to the field of genetic engineering. For example, the protein of the present invention can be used in the field of pharmaceuticals as a therapeutic agent.
A gene is transcribed by forming a transcription initiation complex including RNA polymerase bound to the promoter region upstream to the gene. Gene transcription is considered to be regulated mainly by transcriptional regulatory factors binding to the promoter and interactive with the transcription initiation complex. In general, a transcriptional regulatory factor comprises a DNA binding region and a transcriptional activation region. The transcriptional activation region is considered to be involved in the interaction.
The relation between the expression of specific genes and various diseases has been revealed in the field of medical treatment. For example, the expression of cytokine genes, such as TNF (Molecular Medicine 33: 1010-1020 (1996)), IL-1 (Clin. Immunol. 27: 18-28 (1995)), and IL-8 (Clin. Immunol. 27: 80-85 (1995)), are reported to be associated with various diseases, including inflammation. Hyperactivated transcription of IL-2 is involved in the immune diseases such as graft rejection.
Furthermore, diseases for which there is currently no effective treatment, such as virus infections including cancer and AIDS, can be controlled if expression of a responsible cancer gene (such as c-Myc) and virus gene is inhibited.
Such diseases can be treated if the transcriptional regulatory factor involved in the disease is isolated and its function is controlled. Therefore, transcriptional regulatory factors drew much attention as targets for developing new drugs.
An inhibitor of a transcriptional regulatory factor has been screened by a DNA binding inhibitory experiment in a chemical library or natural substances derived from bacteria or plants, or by drug design based on the structure of a gene or a transcriptional factor to be controlled (Peterson, M. G. et al., Trends Biotechnol. 11: 11-18 (1993).
The DNA binding inhibitory experiment requires screening numerous samples. However, there is no effective method of screening at present.
Furthermore, drug design requires that the structure of transcriptional regulatory factors must be known in detail. Therefore, its application is limited.
An objective of the present invention is to provide a transcriptional regulatory factor having transcription-inhibitory activity, more specifically, a transcriptional regulatory factor having transcription-inhibitory activity on a specific promoter.
Normal animal cells contain hypoxanthine-guanine phosphoribosyl-transferase (HGPRT), thus, uptake 6-thioguanine (6-TG) in their nucleic acid synthetic pathway. Those cells die in a 6-TG-containing medium due to its toxicity. In contrast, HGPRT-deficient mutant cells survive and proliferate in the 6-TG-containing medium, unaffected by the toxicity because those cells do not uptake 6-thioguanine (6-TG) in the nucleic acid synthetic pathway. Therefore, in the HGPRT-deficient mutant cells, the survival of the cells in the presence of 6-thioguanine (6-TG) can be determined by controlling the expression of HGPRT (or a protein with a similar function). The present inventors constructed the system to search for a gene involved in intracellular signal transduction, using the above characteristics of the HGPRT-deficient mutant cells.
It is evident that a promoter of the interleukin 8 gene (IL-8) is activated in response to stimulation by a tumor necrosis factor (TNF). The present inventors prepared an expression plasmid carrying a xanthine-guanine phosphoribosyl-transferase (gpt) gene which causes cell death when expressed in the presence of 6-TG similarly to HGPRT, by inserting the gene downstream to the IL-8 promoter sequence in the plasmid vector, and introduced the resultant plasmid into HGPRT-deficient cells. The cells die in the presence of a suitable quantity of 6-TG in response to the TNF stimulation because of the expression of gpt gene. However, when a certain gene capable of inhibiting the signal transduction pathway from the TNF stimulation to the activation of the IL-8 promoter is introduced into the above cells, the expression of gpt can be inhibited and then the cells survive. Therefore, genes capable of inhibiting the intracellular signal transduction can be isolated by introducing various genes in said cells and selecting surviving cells.
The present inventors successfully isolated four genes (S1-15, S1-b2, S2-3, and S20-1) which were suppressive for IL-8 promoter activation from a cDNA library by using the above screening system. The present inventors specifically analyzed xe2x80x9cS1-15xe2x80x9d which consistently exhibited IL-8 promoter inhibitory activity.
First, the present inventors determined the sequence of xe2x80x9cS1-15.xe2x80x9d Homology searches based on the determined nucleotide sequence found two homologous genes which were reported as those encoding transcriptional regulatory factors.
The present inventors noticed that the transcriptional regulatory factors, which were supposed to enhance transcription inherently, were in fact inhibitory for IL-8 promoter. Then, the present inventors analyzed the amino acid sequence of S1-15 in more detail. They found that S1-15 comprised a DNA binding region within the above transcriptional regulatory factor but lacked most of the rest, including the regions that interact with other factors involved in transcription.
Finally, the present inventors found that the transcriptional regulatory factors can be modified into transcriptional inhibitory factors by deleting sequences other than regions having DNA binding activity of transcriptional regulatory factors.
Based on the fact that S1-15 inhibits the activation of IL-18 promoter, the present inventors subsequently analyzed whether S1-15 has anti-inflammatory activity. The result demonstrated that S1-15 strongly inhibits the human IL-8 production stimulated with IL-1 xcex2 in MRC-5 cells. It also inhibited the production by IL-1 xcex2-stimulated MRC cells of a neutrophil chemotactic factor GRO xcex1 at the level comparable to the inhibition of IL-8 production. Thus, the present inventors have found that S1-15 is a protein having a potent anti-inflammatory activity.
Specifically, the present invention relates to:
(1) a protein having transcription inhibitory activity and lacking at least a part of regions other than a region having DNA binding activity in a transcriptional regulatory factor,
(2) the protein of (1), wherein said protein lacks at least a part of the region interacting with other factors involved in transcription in the transcriptional regulatory factor,
(3) a protein having inhibitory activity on transcription of interleukin 8 gene and having the amino acid sequence of SEQ ID NO: 1, or said amino acid sequence in which one or more amino acid residues are substituted, deleted, or added,
(4) a protein having the amino acid sequence of SEQ ID NO: 1,
(5) a DNA encoding the protein of (1) to (4),
(6) a vector comprising the DNA of (5),
(7) a cell carrying the vector of (6),
(8) a method of inhibiting transcription of a specific gene, which comprises introducing the protein of (1) or (2) into cells,
(9) a method of inhibiting transcription of the interleukin 8 gene, which comprises introducing the protein of (3) or (4) into cells,
(10) a method of inhibiting transcription of a specific gene, which comprises introducing a vector comprising DNA encoding the protein of (1) or (2) into cells and allowing the protein of (1) or (2) to be expressed in the cells,
(11) a method of inhibiting transcription of the interleukin 8 gene, which comprises introducing a vector comprising DNA encoding the protein of (3) or (4) into cells and allowing the protein of (3) or (4) to be expressed in the cells.
The invention also includes a substantially pure polypeptide comprising (1) an amino acid sequence at least 60% (e.g., 70%, 80%, 90%, 95%, or 99%) identical to SEQ ID NO: 1, (2) an amino acid sequence that is SEQ ID NO: 1 containing at least one conservative amino acid substitution (preferably between 1-30, and more preferably 15 or fewer (e.g., 5 or fewer or even 3 or fewer) substitutions), or (3) encoded by a first nucleic acid consisting of SEQ ID NO: 1. The polypeptide can inhibit transcription of interleukin 8.
The invention also features an isolated nucleic acid encoding a polypeptide of the invention.
The invention further features vectors and transformed host cells, containing a nucleic acid of the invention.
Also features in the invention is a method of inhibiting transcription of the interleukin 8 gene, the method comprising introducing into cells a substantially pure polypeptide comprising (1) an amino acid sequence at least 60% (e.g., 70%, 80%, 90%, 95%, or 99%) identical to SEQ ID NO: 1, (2) an amino acid sequence that is SEQ ID NO: 1 containing at least one conservative amino acid substitution (preferably between 1-30, and more preferably 15 or fewer (e.g., 5 or fewer or even 3 or fewer) substitutions), or (3) encoded by a first nucleic acid that hybridizes under stringent conditions to a second nucleic acid consisting of SEQ ID NO: 1. The polypeptide can inhibit transcription of interleukin 8. Other methods included in the invention include a method of inhibiting transcription of the interleukin 8 gene, the method comprising introducing a vector containing a nucleic acid of the invention into cells and allowing a polypeptide of the invention to be expressed in the cells.
The invention also features a pharmaceutical composition comprising a polypeptide or a nucleic acid of the invention in an anti-inflammatory effective amount and a pharmaceutically acceptable carrier.
The term xe2x80x9ca transcriptional regulatory factorxe2x80x9d used herein means a protein controlling transcription of a gene, other than basal transcription factors. The term xe2x80x9ca basal transcription factorxe2x80x9d means a factor which forms a transcription initiation complex with RNA polymerase and DNA.
The term xe2x80x9ca factor involved in transcriptionxe2x80x9d used herein means a factor regulating gene transcription, including basal transcription factors.
The term xe2x80x9csubstantially purexe2x80x9d used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological compounds, such as those in cellular material, viral material, or culture medium, with which the polypeptide may have been associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). The substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
An xe2x80x9cisolated nucleic acidxe2x80x9d: is a nucleic acid which has a non-naturally occurring sequence, or which has the sequence of part or all of a naturally occurring gene but is free of the gene that flank the naturally occurring gene of interest in the genome of the organism in which the gene of interest naturally occurs. The term therefore includes a recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote. It also includes a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment. It also includes a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are mixtures of DNA molecules, vectors, or clones as they occur in a DNA library such as a cDNA or genomic DNA library. Also excluded are RNA molecules that consist of naturally-occurring sequences (e.g., naturally-occurring mRNA) except where the RNA is in a purified state such that it is at least 90% free of other naturally-occurring RNA species. Thus, a naturally-occurring mRNA in a whole mRNA preparation prepared from a cell would not be an xe2x80x9cisolated nucleic acid,xe2x80x9d but a single mRNA species purified to 90% homogeneity from that whole mRNA preparation would be.
As used herein, xe2x80x9cpercent identityxe2x80x9d of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990) modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a reference polypeptide (e.g., SEQ IS NO: 2). To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the retrospective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.
A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which the amino acid residue is replaced with another residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acid with basic side chains (e.g., lysine, glutamic acid), uncharged polar side chains (e.g., glycine, aspargine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
By xe2x80x9chybridizes under stringent conditionsxe2x80x9d is meant specific and non-covalent equilibrium binding by base-pairing to an immobilized reference nucleic acid in a hybridization solution containing 0.2xc3x97SSC (1.75 g/l NaCl, 0.88 g/l Na3citrate 2H2O;pH 7.0) and 0.1% (w/v) sodium dedecylsulfate at 68xc2x0 C. Washings, if any are required to achieve equilibrium, are carried out with the hybridization solution.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions, will control. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
First, the present invention relates to a protein having transcription inhibitory activity and lacking at least a part of the sequences other than those having DNA binding activity of transcriptional regulatory factors.
The protein of the present invention generally includes the region having DNA binding activity of transcriptional regulatory factors. A region having DNA binding activity of transcriptional regulatory factors varies depending on the transcriptional regulatory factor. For example, the bzip (basic-leucine zipper) region in c-JUN (Sutherland, J. A. et al., Genes Dev., 6: 1810-1819(1992)) and Zn finger region in Sp1 (Kadonaga, J. T. et al., Science 242: 1556-1570 (1988). The region having DNA binding activity of transcriptional regulatory factors can vary depending on the upstream region of the gene to be bound. The region having DNA binding activity of transcriptional regulatory factors can be determined by preparing various deletion mutants of transcriptional regulatory factor genes and detecting the DNA binding activity using gel shift assay.
The protein of the present invention also possesses transcription inhibitory activity and lacks at least a part of regions other than those having DNA binding activity. The sequence other than the region having DNA binding activity is not particularly limited as long as deletion of the sequence imparts transcription inhibitory activity. This region preferably interacts with other factors involved in transcription, more preferably, a transcriptional activation region (a region interacting with basal transcription factors). The transcriptional activation region can be determined by replacing amino acid residues by site-specific mutagenesis and detecting the decreased transcription activity by the replacement (Gill, G. et al., Cell 51: 121-126 (1987)).
The protein of the present invention can include not only an amino acid sequence of a part of natural transcriptional factors but also a sequence in which one or more amino acids are deleted, substituted, or added, as long as the protein has the transcription inhibitory activity. Such deletion, substitution, or addition of amino acids can be performed by site-specific mutagenesis well known in the art at the time this application was filed (Nucleic Acid Res., 10: pp. 6487).
The protein can be prepared by the method described below.
First, if a gene""s transcription is to be inhibited but the gene encoding the transcriptional regulatory factor is unknown, the gene encoding the transcriptional regulatory factor should be isolated. cDNA encoding the protein capable of binding to the transcriptional regulatory region can be isolated, for example, by screening a cDNA library inserted in a phage vector using the transcriptional regulatory region of a gene whose transcription is to be inhibited as a probe (Vinson, C. R. et al., Genes Dev. 2: 801-806 (1988)).
Second, at least a part of regions other than those having DNA binding activity in the isolated gene encoding the transcriptional regulatory factor are deleted. This deletion is enabled by creating new restriction sites by introducing point mutation in the DNA sequence and utilizing the restriction sites using recombinant DNA technology (commonly used genetic engineering technology such as methods described in xe2x80x9cMolecular cloning (Sambrook, J et al., Molecular Cloning; A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, New York (1989),xe2x80x9d xe2x80x9cLaboratory Manual of Genetic Engineering, Muramatsu, M. ed., Maruzen (1989)xe2x80x9d) or the xe2x80x9cKunkel methodxe2x80x9d (Methods Enzymol. 85: 2763-2766 (1988)). The deletion also can be made by PCR (Higuch, R. PCR Protocols, Academic press, INC. 177-183 (1990)).
Third, the obtained DNA is ligated with an appropriate vector, and the vector is introduced into cells to detect transcription inhibitory activity of the expressed protein. The vector to be used is not particularly limited. Preferable examples of the vector include xe2x80x9cpcDL-SRxcex1296xe2x80x9d comprising SRxcex1 promoter capable of effectively expressing the gene to be expressed (Takebe, Y. et al., Mol. Cell. Biol. 8: 466-472 (1988)), xe2x80x9cpEF-BOSxe2x80x9d comprising the promoter of the elongation factor (Mizushima, S. Nucleic Acid Res. 18 (1990)), or xe2x80x9cpCAGGSxe2x80x9d comprising CAG (cytomegalovirus IE enhancer+chicken xcex2-globin poly(A) signal) promoter (Niwa, H. et al., GENE 108: 193-200 (1990)).
The cells into which the vector is introduced are not particularly limited. xe2x80x9cMRC-5 SV1 TG1 cellxe2x80x9d (Riken Cell Bank), xe2x80x9cVA-13 cellxe2x80x9d (Riken Cell Bank), or xe2x80x9cRERF-LC-AI cellxe2x80x9d (Riken Cell Bank) is preferable.
The vector can be introduced into the cells by the DEAE-dextran method (Luthman, H. et al., Nucleic Acids Res. 11: 1295-1308 (1983)), the calcium phosphate method (Graham, F. L. et al., Virology 52: 456-457. (1973)), electroporation (Neumann, E. et al., EMBO J. 1: 841-845 (1982), or similar methods.
The transcriptional inhibitory activity can be detected by, for example, reporter assay (Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, N.Y. (1989)).
Fourth, the protein whose transcription inhibitory activity was detected is isolated and purified. The isolation and purification can be performed by inserting a gene encoding the protein whose transcription inhibitory activity was detected into an expression vector, transforming prokaryotic or eukaryotic host cells with said vector, and purifying the expressed protein by chromatography or a similar method. For example, when E. coli is used as a host, the method of Smith et al. (Smith, D. B. et al., Current Protocols in Molecular Biology Vol. 2, John Wiley and Sons, New York, 16.7. (1990)) can be used.
The protein of the-present invention can be prepared by the methods described above.
The present invention also relates to a method of inhibiting transcription of a specific gene using a protein having transcriptional inhibitory activity and lacking at least a part of regions other than those having DNA binding activity of transcriptional regulatory factors.
One embodiment relates to the method of inhibiting transcription of a specific gene and is characterized by introducing said protein into cells.
The cells into which the protein of this invention is to be introduced are not particularly limited. The protein of the present invention can be introduced into cells by microinjection, electroporation, the liposomal method, the erythrocyte ghost method, or some similar method.
Another embodiment relates to a method of inhibiting transcription of a specific gene and is characterized by introducing a vector comprising DNA encoding the protein of the present invention into cells and allowing said protein to be expressed in cells.
Any vector can be used as long as it can express the inserted gene. When animal cells are used, vectors such as xe2x80x9cpcDL-SRxcex1296xe2x80x9d comprising SRQ promoter capable of effectively expressing a gene to be expressed (Takebe, Y. et al., Mol. Cell. Biol. 8: 466-472 (1988)), xe2x80x9cpEF-BOSxe2x80x9d comprising the promoter of the elongation factor (Mizushima, S. Nucleic Acid Res. 18: (1990)), and xe2x80x9cpCAGGSxe2x80x9d comprising CAG (cytomegalovirus IE enhancer+chicken xcex2-globin poly(A) signal) promoter (Niwa, H. et al., GENE 108: 193-200 (1990)) are preferable.
The vector can be introduced into cells by the DEAE-dextran method (Luthman, H. et al., Nucleic Acids Res. 11: 1295-1308 (1983)), the calcium phosphate method (Graham, F. L. et al., Virology 52: 456-457. (1973)), electroporation (Neumann, E. et al., EMBO J. 1: 841-845 (1982), or a similar method.
When expression of a gene whose transcription is controlled by the protein of the present invention correlates with a disease, the protein is particularly useful as a therapeutic agent for the disease. For example, if a specific gene is revealed to be an oncogene, the transcriptional regulatory factor based on the sequence upstream of the oncogene is isolated. The transcriptional regulatory factor gene is then isolated to prepare the protein of the present invention. In this case, the protein of the present invention can be used as an anticancer agent. The protein of the present invention can be administered to a patient by subcutaneous injection or other suitable method.
Furthermore, the protein of the present invention can be used to inhibit a disease by expressing it in the patient""s body. Transcriptional regulatory factors are believed to bind to the upstream portion of a specific gene with high specificity to the gene. Therefore, the protein of the present invention derived from the transcriptional regulatory factor can also have high specificity to the gene and thus be useful in the field of gene therapy. In this case, the gene encoding the protein of the present invention is inserted into an appropriate vector and can be introduced into the body by, for example, a method using a virus vector or a method using membrane-fused liposomes. The virus vector to be used is not particularly limited. Preferable examples thereof include retrovirus vectors, adenovirus vectors, AAV (adeno-associated virus) vectors, herpesvirus vectors, and HIV vectors.
IL-8 is reported to be produced in various inflammatory diseases, such as chronic articular rheumatism, gouty arthritis, psoriasis, contact dermatitis, sepsis, cataplectic pulmonary fibrosis, adult respiratory distress syndrome, inflammatory enteropathy, immune angiitis, glomerulonephritis, urinary tract infection, myocardinal infarction, respiratory tract infection, asthma, perinatal infection, and rejection to transplanted organs (Matushima, K. et al., Chem Immunol 51: 236-265 (1992)). The protein inhibiting transcription of IL-8 and having the sequence of SEQ ID NO: 1 can be used as a therapeutic agent for the above-described diseases.
In fact, S1-15 protein, which was found by the present inventors, strongly suppresses the production of human IL-8 and Gro-xcex1 in MRC-5 cells, indicating that it has anti-inflammatory activity. Thus, S1-15 protein (the protein having the sequence of SEQ ID: 1 or its functionally equivalent derivatives) or DNA encoding the protein can be used for the treatment of the diseases.
When S1-15 protein is used as an anti-inflammatory agent, it can be formulated with a pharmaceutically acceptable carrier of medium (e.g., physiological saline, vegetable oil, suspension, surfactant, stabilizer, and the like) by the known methods and administered to patients. The preparation is administered through a suitable route, such as transcutaneous, intranasal, transbronchial, intramuscular, intravenous, or oral administration, depending on the property of the compound. Although the dosage may be varied depending on the age, weight, and condition of the patent, and the method of administration, one skilled in the art can readily select a suitable dosage. When DNA encoding S1-15 protein is used as an anti-inflammatory agent for gene therapy, the DNA can be incorporated into a vector capable of functioning in vivo, and the vector can be administered to patients by the in vivo or ex vivo method. Alternatively, the DNA can be administered to patients without using such a viral vector.